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Audrey E K Hutcheon, Xiaoqing Q Guo, Sriniwas Sriram, Jennifer A Tran, James D Zieske, Audrey E K Hutcheon; p38/TGFβ-signaling pathway involved in induction of thrombospondin-1 in human corneal fibroblasts. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2357.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that competitive inhibition of the SMAD/TGFβ-signaling pathway does not reduce the expression of Thrombospondin-1 (TSP1), a known downstream TGFβ-target protein, in human corneal fibroblasts (HCF). In addition, when HCF were stimulated with TGF-β1 (T1), they were induced to produce more TSP1, as well as, fibrotic markers, cellular fibronectin (cFN) and collagen type III (Col III). Therefore, in this study, we hypothesized that an alternate TGFβ-signaling pathway—the p38 pathway—induces TSP1 in HCF, and may also affect cFN and Col III.
Human corneal fibroblasts (HCF) were grown in EMEM + 10% FBS until they were 60-70% confluent, at which time the cells were cultured overnight in EMEM only, to serum starve the cells. The next day, HCF were cultured as follows for 24 hours: 1) Control: EMEM only; 2) T1: EMEM + 2ng/ml T1; 3) p38inh: EMEM + 10μM of p38 inhibitor (SB202190); and 4) Both: EMEM + 2ng/ml T1 + 10μM p38 inhibitor. Cells then were harvested and processed for western blot or quantitative real-time polymerase chain reaction (qRT-PCR) for TSP1, cFN, and Col III. Experiments were repeated at least three times.
TSP1 protein and mRNA is present in serum starved HCF (control), and as expected, upon treating these cells with T1, TSP1 protein increased; however, the mRNA remained relatively similar to control. In the p38inh samples, both TSP1 protein and mRNA dramatically decreased when compared with both the control and T1 samples. When both T1 and p38 inhibitor were introduced, TSP1 mRNA levels dramatically decreased, similar to the p38inh samples; however, TSP1 protein levels only decreased compared to T1 samples. Col III and cFN responded in a similar manner as TSP1.
Interestingly, the p38 inhibitor did decrease the protein and/or mRNA levels of TSP1, Col III, and cFN in HCF, thus showing that in HCF these proteins are downstream from the p38/TGFβ-signaling pathway. Also, the discrepancy between mRNA and protein levels for TSP1 suggests that there are other factors involved in either the stabilization or degradation of TSP1 in HCF.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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